Chondrocyte Cluster Formation Research Articles

Development of alginate-collagen interpenetrating network for osteoarthritic cartilage by in situ softening

This study presents an alginate-collagen interpenetrating network (IPN) matrix of incorporating collagen fibrils into an alginate hydrogel by physical mixing and controlled gelation. The resulting matrix closely mimics the physiological and pathological stiffness range of the chondrocyte pericellular matrix (PCM). Chondrocytes were cultured within three-dimensional (3D) alginate-collagen IPN matrices with varying stiffness, namely Firm, Medium, and Soft. Alginate lyase was introduced to study the effects of the changes in stiffness of the Firm on chondrocyte response by in situ softening. The developed alginate-collagen IPN matrix displayed good cell-biocompatibility. Compared with stiffer tissue culture plastic (TCP), chondrocytes grown within Firm displayed a stabilized differentiated phenotype characterized by higher expression levels of aggrecan, collagen II, and SOX-9. Moreover, the developed alginate-collagen IPN matrix exhibited a gradually increased percentage of propidium iodide (PI)-positive dead cells with decreasing stiffness. Softer matrices directed cells towards higher proliferation rates and spherical morphologies while stimulating chondrocyte cluster formation. Furthermore, reducing Firm stiffness by in situ softening decreased aggrecan expression, contributing to matrix degradation similar to that seen in osteoarthritis (OA). Hence, the 3D alginate-collagen IPN constructs hold significant potential for in vitro replicating PCM stiffness changes observed in OA cartilage.

Bidirectionally validated in silico and in vitro formation of specific depth zone-derived chondrocyte spheroids and clusters.

3D multicellular self-organized cluster models, e.g., organoids are promising tools for developing new therapeutic modalities including gene and cell therapies, pharmacological mechanistic and screening assays. Various applications of these models have been used extensively for decades, however, the mechanisms of cluster formation, maintenance, and degradation of these models are not even known over in-vitro-life-time. To explore such advantageous models mimicking native tissues or organs, it is necessary to understand aforementioned mechanisms. Herein, we intend to clarify the mechanisms of the formation of cell clusters. We previously demonstrated that primary chondrocytes isolated from distinct longitudinal depth zones in articular cartilage formed zone-specific spherical multicellular clusters in vitro. To elucidate the mechanisms of such cluster formation, we simulated it using the computational Cellular Potts Model with parameters were translated from gene expression levels and histological characteristics corresponding to interactions between cell and extracellular matrix. This simulation in silico was validated morphologically with cluster formation in vitro and vice versa. Since zone specific chondrocyte cluster models in silico showed similarity with corresponding in vitro model, the in silico has a potential to be used for prediction of the 3D multicellular in vitro models used for development, disease, and therapeutic models.

Antigen-induced arthritis of the temporomandibular joint via repeated injections of bovine serum albumin in domestic pigs

Rheumatoid arthritis (RA) is a chronic inflammatory disease and the temporomandibular joint (TMJ) is affected in up to 50%, resulting in pain, limited mouth opening and dental malocclusion. The outcome of conservative and surgical therapies is unsatisfying in many cases. The purpose of this study was to establish a large animal model of antigen-induced arthritis (AIA) of the TMJ that enables the investigation of the pathogenesis of RA and the evaluation of new therapies. In five domestic pigs, systemic immunization was performed via consecutive intramuscular injections of bovine serum albumin (BSA). Then, AIA was induced via the application of BSA into the TMJ. Injection with saline served as the control. After ten weeks, the joints and adjacent tissues were harvested for histological analysis and cytokine quantification. The changes observed in the AIA specimens included severe synovial inflammation, cartilage-specific glycosaminoglycan content loss, and cartilage surface and discus alterations as well as the formation of chondrocyte clusters. Protein analyses of the synovia showed enhanced levels of IL-1β, IL-6, TNFα and VEGF. A porcine model of immunologic arthritis of the TMJ was successfully established. This model may be used in future studies to investigate the underlying pathogenesis of RA and new therapeutic strategies.

Blocking of matrix metalloproteinases-13 responsive peptide in poly(urethane urea) for potential cartilage tissue engineering applications.

The matching of scaffold degradation rate with neotissue growth is required for tissue engineering applications. Timely provision of proper spaces especially for cartilage tissue engineering plays a pivotal role in chondrocyte cluster formation. In this study, poly(urethane urea) was synthesized using conventional two-stage method by extending the isocyanate group terminated prepolymers with different amounts of GPLGLWARK peptide, which responses the degrading induced by matrix metalloproteinase 13, the main proteinase for cartilage matrix degradation. The Fourier transform infrared spectrometer with the attenuated total reflection and 1H nuclear magnetic resonance spectra revealed that the peptides were introduced to poly(urethane urea) according to the characteristic absorption bands of the peptide and the newly formed urea bonds. The ultraviolet-visible spectroscopy spectra showed that the weight percentages of the peptide in the three poly(urethane urea) were 25%, 32%, and 35%. Atomic force microscopy images revealed that phase separation occurred in all poly(urethane urea) samples and became increasingly apparent with increasing amount of peptides introduced. Mechanical tests showed that the poly(urethane urea) strength increased with increasing amount of peptides in poly(urethane urea). Poly(urethane urea) proteolysis in matrix metalloproteinase 13 solution was more rapid than hydrolysis in aqueous buffer, and proteolysis rate was dependent on the amount of peptides in poly(urethane urea). Cell proliferation on the material surface in vitro displayed nontoxicity for all synthesized poly(urethane urea). In vivo subcutaneous implantation evaluation revealed the presence of local foreign body reactions triggered by poly(urethane urea) but was not due to peptide in poly(urethane urea). Moreover, the synthesized poly(urethane urea) with significant phase separation did not degrade under the matrix metalloproteinase 13 free subcutaneous environment, but poly(urethane urea) with minimal phase separation was degraded by attacking of the enzymes adsorbed on the hydrophobic surface through non-specific adsorption.

Promoting chondrocyte cell clustering through tuning of a poly(ethylene glycol)-poly(peptide) thermosensitive hydrogel with distinctive microarchitecture

Hydrogels are considered to be attractive cell-matrix for chondrocytes due to their similarity in properties to the natural cartilage. However, the formation of chondrocyte cell clusters in hydrogels has been mostly limited to naturally-derived or relatively fast degrading materials. In this study, a series of diblock copolymer poly(ethylene glycol)-poly(alanine) (mPEG-PA) was synthesized and investigated as injectable biomimic hydrogels for the culturing of chondrocytes. Depending on the poly(alanine) chain length, afforded hydrogels exhibited variable mechanical property and microarchitecture due to difference in secondary structure arrangement. After 21days of culture, cell clusters were observed in all hydrogels with longer PA chains and these hydrogels supported more homogenous and established clustering as well as significantly higher glycosaminoglycan and collagen deposition. Interestingly, scanning electron microscopy revealed a distinct micron range fibrillar-like microarchitecture that may be responsible for maintaining chondrocyte phenotype and matrix production. In addition, micrographs revealed the presence of collagen fibrils and an extensive extracellular matrix network. Therefore, it is reasonable to conclude that mPEG-PA hydrogels possess the desirable properties for chondrocyte cluster formation and serve as potential candidate in cartilage tissue engineering.

HISTOLOGICAL MARKERS OF DEGENERATION AND REGENERATION OF THE HUMAN INTERVERTEBRAL DISK

ABSTRACT Objective: To define histological scores for intervertebral disc degeneration that would enable the definition of morphological characteristics of disease, besides improving knowledge of the lumbar degenerative disc disease by means of immunohistochemical markers. Methods: Hematoxylin and Eosin, Alcian/PAS, Masson Trichrome and Safranin O/FCF staining was used on the intervertebral disc degeneration sections of patients with lumbar degenerative disc disease. The protein markers defined in immunohistochemistry were cell proliferation (Ki-67) and apoptosis (p53). Results: The study data enabled the determination of Safranin O/FCF stain as the most effective one for evaluating parameters such as area, diameter, and number of chondrocyte clusters. The importance of using stains in association, such as Safranin O/FCF, Masson Trichrome, Alcian/PAS and Hematoxylin and Eosin, was also determined, as they are complementary for the histopathological verification of intervertebral disc degeneration. By expressing proteins using the immunohistochemistry technique, it was possible to consider two stages of disc degeneration: cell proliferation with chondrocyte cluster formation, and induction of apoptosis. Conclusion: This study enabled the histological and immunohistochemical characterization to be determined for lumbar degenerative disc disease, and its degrees of evolution, by determining new disc degeneration scores.

Inhibition of cartilage degradation and suppression of PGE2 and MMPs expression by pomegranate fruit extract in a model of posttraumatic osteoarthritis

ObjectiveOsteoarthritis (OA) is characterized by cartilage degradation in the affected joints. Pomegranate fruit extract (PFE) inhibits cartilage degradation in vitro. The aim of this study was to determine whether oral consumption of PFE inhibits disease progression in rabbits with surgically induced OA. MethodsOA was surgically induced in the tibiofemoral joints of adult New Zealand White rabbits. In one group, animals were fed PFE in water for 8 wk postsurgery. In the second group, animals were fed PFE for 2 wk before surgery and for 8 wk postsurgery. Histologic assessment and scoring of the cartilage was per Osteoarthritis Research Society International guidelines. Gene expression and matrix metalloproteinases (MMP) activity were determined using quantitative reverse transcriptase polymerase chain reaction and fluorometric assay, respectively. Interleukin (IL)-1 β, MMP-13, IL-6, prostaglandin (PG)E2, and type II collagen (COL2A1) levels in synovial fluid/plasma/culture media were quantified using enzyme-linked immunosorbent assay. Expression of active caspase-3 and poly (ADP-ribose) polymerase p85 was determined by immunohistochemistry. Effect of PFE and inhibitors of MMP-13, mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB was studied in IL-1 β-stimulated rabbit articular chondrocytes. ResultsSafranin-O-staining and chondrocyte cluster formation was significantly reduced in the anterior cruciate ligament transaction plus PFE fed groups. Expression of MMP-3, MMP-9, and MMP-13 mRNA was higher in the cartilage of rabbits given water alone but was significantly lower in the animals fed PFE. PFE-fed rabbits had lower IL-6, MMP-13, and PGE2 levels in the synovial fluid and plasma, respectively, and showed higher expression of aggrecan and COL2A1 mRNA. Significantly higher numbers of chondrocytes were positive for markers of apoptosis in the joints of rabbits with OA given water only compared with those in the PFE-fed groups. PFE pretreatment significantly reduced IL-1 β induced IL-6 and MMPs expression in rabbit articular chondrocytes. These effects were also mimicked using MMP-13, MAPK, and NF-κB inhibitors in IL-1 β-stimulated rabbit chondrocytes. In an in vitro activity assay, PFE blocked the activity of MMP-13. Like MAPK and NF-κB inhibitors, PFE was also effective in inhibiting IL-1 β-induced PGE2 production in rabbit chondrocytes. PFE also reversed the inhibitory effect of IL-1β on COL2A1 mRNA and protein expression in IL-1 β-stimulated rabbit chondrocytes. ConclusionThe present data highlight the chondroprotective effects of PFE oral consumption in a model of posttraumatic OA and suggest that PFE-derived compounds may have potential value in the management of OA.

Anti-inflammatory effect of dialysable leucocyte extract in a rat model of osteoarthritis: histopathological and molecular characterization

Objectives: To evaluate the effect of dialysable leucocyte extract (DLE) on pro- and anti-inflammatory profiles in a rat model of osteoarthritis (OA).Method: Forty-eight male Wistar rats were divided into three groups: normal rats without treatment, OA rats treated with placebo, and OA rats treated with DLE. After treatment, the animals were killed to obtain cartilage for histological analysis and to determine the expression of pro- and anti-inflammatory cytokines by reverse transcription multiplex polymerase chain reaction (RT-MPCR) and immunohistofluorescence analyses.Results: Histological analysis revealed that OA cartilage from rats treated with DLE displayed similar characteristics to non-OA cartilage from the control group. The OA cartilage treated with placebo showed alterations in the cellular architecture and in chondrocyte cluster formation. Analysis of cytokine expression by RT-MPCR showed that OA cartilage from DLE-treated rats expressed platelet-derived growth factor (PDGF), interferon (IFN)-γ, and fibroblast growth factor (FGF)-2, similar to non-OA cartilage from the control group. However, OA cartilage from rats treated with placebo expressed interleukin (IL)-1, PDGF, and I kappa B (IκB). Confocal immunodetection of FGF-2, PDGF, and non-phosphorylated IκB showed that they were distributed in the cytoplasm of most chondrocytes in OA cartilage from DLE-treated rats whereas no nuclear factor kappa B (NF-κB) expression was observed in the nuclei. Instead, in OA cartilage from the placebo group, only weak FGF-2 staining was observed, PDGF and IκB were not detected, and NF-κB was strongly observed in both cytoplasm and nuclei.Conclusions: Our findings suggest that DLE treatment modifies the OA process, promoting the expression of anti-inflammatory cytokines and diminishing the inflammatory effects, avoiding the nuclear translocation of NF-κB in chondrocytes.

Effect of Laminin-A4 inhibition on cluster formation of human osteoarthritic chondrocytes.

Formation of chondrocyte clusters is not only a morphological sign of osteoarthritis but it is also observed in cell culture. Active locomotion of chondrocytes is controlled by integrins in vitro. Integrins bind to Laminin-A4 (LAMA4), a protein that is highly expressed in vivo in clusters of hypertrophic chondrocytes. We tested if LAMA4 is relevant for cluster formation. Human chondrocytes were cultured in a 2D matrigel model and treated with different concentrations of a monoclonal inhibitory anti-LAMA4-antibody. Migration and cluster formation was analysed using live cell imaging technique. Full genome gene expression analysis was performed to assess the effect of LAMA4 inhibition. The data set were screened for genes relevant to cell motility. F-actin staining was performed to document cytoskeletal changes. Anti-LAMA4 treatment significantly reduced the rate of cluster formation in human chondrocytes. Cells changed their surface morphology and exhibited fewer protrusions. Expression of genes associated with cellular motility and migration was affected by anti-LAMA4 treatment. LAMA4-integrin signalling affects chondrocyte morphology and gene expression in vitro, thereby contributing to cluster formation in human osteoarthritic chondrocytes.

Menisectomized miniature Vietnamese pigs develop articular cartilage pathology resembling osteoarthritis

Animal models have been used to understand the basic biology of osteoarthritis (OA) and have helped to identify new candidate biomarkers for the early diagnosis and treatment of this condition. Small animals cannot sufficiently mimic human diseases; therefore, large animal models are needed. Pigs have been used as models for human diseases because they are similar to humans in terms of their anatomy, physiology and genome. Hence, we analyzed articular cartilage and synovial membrane pathology in miniature Vietnamese pigs after a unilateral partial menisectomy and 20-day exercise regimen to determine if the pigs developed pathological characteristics similar to human OA. Histological and protein expression analysis of articular cartilage from menisectomized pigs revealed the following pathologic changes resembling OA: fibrillation, fissures, chondrocyte cluster formation, decrease in proteoglycan content and upregulation of the OA-associated proteins MMP-3, MMP-13, procaspase-3 and IL-1β. Moreover, histological analysis of synovial membrane revealed mild synovitis, characterized by hyperplasia, cell infiltration and neoangiogenesis. Pathological changes were not observed in the contralateral joints or the joints of sham-operated pigs. Further studies are required to validate such an OA model; however, our results can encourage the use of pigs to study early stages of OA physiopathology. Based on their similarities to humans, pigs may be useful for preclinical studies to identify new candidate biomarkers and novel treatments for OA.

CCN1 (Cyr61) Is Overexpressed in Human Osteoarthritic Cartilage and Inhibits ADAMTS-4 (Aggrecanase 1) Activity.

ADAMTS-4, also called aggrecanase 1, is considered to play a key role in aggrecan degradation in human osteoarthritic (OA) cartilage, but information about regulators of ADAMTS-4 aggrecanase activity remains limited. We undertook this study to search for molecules that modulate ADAMTS-4 activity. Molecules copurified with ADAMTS-4 from ADAMTS-4-transfected chondrocytic cells were sequenced by nanoscale liquid chromatography tandem mass spectrometry. Binding activity was determined by immunoprecipitation and solid-phase binding assay. Effects on ADAMTS-4 activity were examined by aggrecan digestion assay. Expression of the binding molecule in OA cartilage and chondrocytes was examined by immunohistochemistry and reverse transcription-polymerase chain reaction. We identified CCN1 (Cyr61) as an ADAMTS-4-binding protein and showed specific binding to the ADAMTS-4 cysteine-rich domain. Aggrecanase activity of ADAMTS-4 was inhibited by interaction with CCN1. Expression of messenger RNA for CCN1 was significantly higher in human OA cartilage than in normal cartilage. CCN1 was immunolocalized to chondrocytes in OA cartilage, showing direct correlations of immunoreactivity with the Mankin score of cartilage lesions and chondrocyte cloning. CCN1 and ADAMTS-4 were commonly coexpressed in clustered chondrocytes. CCN1 expression in OA chondrocytes was down-regulated by interleukin-1α (IL-1α) and up-regulated by transforming growth factor β (TGFβ). ADAMTS-4 expression was induced by treatment with IL-1α or TGFβ, but aggrecanase activity was detected only under stimulation with IL-1α. TGFβ-treated chondrocytes exhibited aggrecanase activity when CCN1 expression was knocked down. Our findings provide the first evidence that CCN1 suppresses ADAMTS-4 activity and that CCN1 overexpression is directly correlated with chondrocyte cloning in OA cartilage. Our results suggest that the TGFβ/CCN1 axis plays a role in chondrocyte cluster formation through inhibition of ADAMTS-4.

Hyaluronic acid injections protect patellar tendon from detraining-associated damage.

Having previously demonstrated that detraining affects patellar tendon (PT) proteoglycan content and collagen fiber organization, we undertook the present study with two aims: to improve knowledge on the adaptation of PT and its enthesis to detraining from a histological and histomorphometric point of view, and to investigate the hypothesis that repeated peri-patellar injections of hyaluronic acid (HA) on detrained PT may reduce and limit detrained associated-damage. Twenty-four male Sprague-Dawley rats were divided into 3 groups: Untrained (n=6), Trained (n=6) (10 wks-treadmill) and Detrained (n=12). In the detrained rats, the left tendon was untreated while the right tendon received repeated peri-patellar injections of either HA or saline (NaCl). Structure and morphology of PTs (modified Movin score, tear density, collagen type I and III) and enthesis (cell morphology, chondrocyte cluster formation, tidemark integrity, matrix staining and vascularization) were evaluated. The left PT and enthesis of the Detrained groups showed altered structure and morphology with the highest Movin score values, the highest percentage of collagen III and the lowest of collagen I; the lowest score values were observed in the Trained and Detrained-HA groups. Detrained-NaCl PTs showed the highest collagen III and the lowest collagen I values with respect to Detrained-HA PTs. This study strengthens previously published data showing the alteration in tendon and enthesis morphology due to discontinuation of training, and provides new data showing that treatment with HA is effective in the maintenance of the structural properties of PT and enthesis in Detrained rats. Such beneficial effects could play a significant role in the management of conservative and rehabilitation strategies in athletes that change type, intensity and duration of training.

Lack of Relationship Between Occupational Workload and Microscopic Alterations in Lumbar Intervertebral Disc Disease

Objectives : The study investigated the impact of occupational workloads on disc surgery specimens. We report the relationship between workload and histological features.Methods : Specimens were collected prospectively from patients suffering from lumbar disc prolapse (n=90) or spinal osteochondrosis (n=19). Histomorphology and occupational workload data and histomorphological features were evaluated. Occupational data were collected in a structured, standardized patient interview assessing lifting and carrying loads. In this way the exposure was assessed for each test subject's entire working life up to surgery.Results : There was no association between cumulative workload and histological patterns. In a subgroup of patients with a workload period of 12 months prior to surgery a relevant formation of chondrocyte clusters (p=0.055) was apparent. Chondrocyte cluster formation was found in 83% (n=74) of the prolapse patients and in 58% (n=11) of the osteochondrosis patients (p=0.02). Fibrocyte mediated scar formation was found in 55% of the prolapse patients and in 45% of the spinal stenosis patients. Chondrocyte clusters and their de novo collagen matrix did not integrate biomechanically sufficient with collagen fibers of the disc. Disintegration of clusters from disc matrix and formation of intra-discal sequesters were observed.Conclusion : Matrix degeneration was common but displayed no relationship to occupational workload or other histological features. Scar formation was observed in every second specimen. Regenerative chondrocyte cluster proliferation was a common feature in disc specimens and tended to be associated in patients with a workload one year before surgery.

Effect of oxygen tension on tissue-engineered human nasal septal chondrocytes.

Tissue-engineered nasal septal cartilage may provide a source of autologous tissue for repair of craniofacial defects. Although advances have been made in manipulating the chondrocyte culture environment for production of neocartilage, consensus on the best oxygen tension for in vitro growth of tissue-engineered cartilage has not been reached. The objective of this study was to determine whether in vitro oxygen tension influences chondrocyte expansion and redifferentiation. Proliferation of chondrocytes from 12 patients expanded in monolayer under hypoxic (5% or 10%) or normoxic (21%) oxygen tension was compared over 14 days of culture. The highest performing oxygen level was used for further expansion of the monolayer cultures. At confluency, chondrocytes were redifferentiated by encapsulation in alginate beads and cultured for 14 days under hypoxic (5 or 10%) or normoxic (21%) oxygen tension. Biochemical and histological properties were evaluated. Chondrocyte proliferation in monolayer and redifferentiation in alginate beads were supported by all oxygen tensions tested. Chondrocytes in monolayer culture had increased proliferation at normoxic oxygen tension (p = 0.06), as well as greater accumulation of glycosaminoglycan (GAG) during chondrocyte redifferentiation (p < 0.05). Chondrocytes released from beads cultured under all three oxygen levels showed robust accumulation of GAG and type II collagen with a lower degree of type I collagen immunoreactivity. Finally, formation of chondrocyte clusters was associated with decreasing oxygen tension (p < 0.05). Expansion of human septal chondrocytes in monolayer culture was greatest at normoxic oxygen tension. Both normoxic and hypoxic culture of human septal chondrocytes embedded in alginate beads supported robust extracellular matrix deposition. However, GAG accumulation was significantly enhanced under normoxic culture conditions. Chondrocyte cluster formation was associated with hypoxic oxygen tension.

Cartilage degeneration at symptomatic persistent olecranon physis in adolescent baseball players.

Background. Elbow overuse injuries are common in adolescent baseball players, but symptomatic persistent olecranon physis is rare, and its pathogenesis remains unclear. Purpose. To examine the histopathological and imaging findings of advanced persistent olecranon physis. Methods. The olecranon physes of 2 baseball pitchers, aged 14 and 15 years, were examined by preoperative magnetic resonance imaging (MRI), and surgical specimens were examined histologically. Results. T2-weighted MRI revealed alterations in the intrachondral signal intensity possibly related to collagen degeneration and increased free water content. Histological findings of specimens stained with hematoxylin-eosin showed complete disorganization of the cartilage structure, hypocellularity, chondrocyte cluster formation, and moderately reduced staining. All these findings are hallmarks of osteoarthritis and are suggestive of cartilage degeneration. Conclusion. Growth plate degeneration was evident in advanced cases of symptomatic persistent olecranon physis. These findings contribute to understanding the pathogenesis of this disease.

A discoidin domain receptor 1 knock-out mouse as a novel model for osteoarthritis of the temporomandibular joint.

Discoidin domain receptor 1 (DDR-1)-deficient mice exhibited a high incidence of osteoarthritis (OA) in the temporomandibular joint (TMJ) as early as 9weeks of age. They showed typical histological signs of OA, including surface fissures, loss of proteoglycans, chondrocyte cluster formation, collagen type I upregulation, and atypical collagen fibril arrangements. Chondrocytes isolated from the TMJs of DDR-1-deficient mice maintained their osteoarthritic characteristics when placed in culture. They expressed high levels of runx-2 and collagen type I, as well as low levels of sox-9 and aggrecan. The expression of DDR-2, a key factor in OA, was increased. DDR-1-deficient chondrocytes from the TMJ were positively influenced towards chondrogenesis by a three-dimensional matrix combined with a runx-2 knockdown or stimulation with extracellular matrix components, such as nidogen-2. Therefore, the DDR-1 knock-out mouse can serve as a novel model for temporomandibular disorders, such as OA of the TMJ, and will help to develop new treatment options, particularly those involving tissue regeneration.

Synoviocytes protect cartilage from the effects of injury in vitro

BackgroundIt is well documented that osteoarthritis (OA) can develop following traumatic joint injury and is the leading cause of lameness and subsequent wastage of equine athletes. Although much research of injury induced OA has focused on cartilage, OA is a disease that affects the whole joint organ.MethodsIn this study, we investigated the impact of synovial cells on the progression of an OA phenotype in injured articular cartilage. Injured and control cartilage were cultured in the presence of synoviocytes extracted from normal equine synovium. Synoviocytes and cartilage were evaluated for catabolic and anabolic gene expression. The cartilage was also evaluated histologically for loss of extracellular matrix molecules, chondrocyte cell death and chondrocyte cluster formation.ResultsThe results indicate synoviocytes exert both positive and negative effects on injured cartilage, but ultimately protect injured cartilage from progressing toward an OA phenotype. Synoviocytes cultured in the presence of injured cartilage had significantly reduced expression of aggrecanase 1 and 2 (ADAMTS4 and 5), but also had increased expression of matrix metalloproteinase (MMP) -1 and reduced expression of tissue inhibitor of metalloproteinases 1 (TIMP-1). Injured cartilage cultured with synoviocytes had increased expression of both collagen type 2 and aggrecanase 2. Histologic examination of cartilage indicated that there was a protective effect of synoviocytes on injured cartilage by reducing the incidence of both focal cell loss and chondrocyte cluster formation, two major hallmarks of OA.ConclusionsThese results support the importance of evaluating more than one synovial joint tissue when investigating injury induced OA.

Development of an in vitro model of injury-induced osteoarthritis in cartilage explants from adult horses through application of single-impact compressive overload

To develop an in vitro model of cartilage injury in full-thickness equine cartilage specimens that can be used to simulate in vivo disease and evaluate treatment efficacy. 15 full-thickness cartilage explants from the trochlear ridges of the distal aspect of the femur from each of 6 adult horses that had died from reasons unrelated to the musculoskeletal system. To simulate injury, cartilage explants were subjected to single-impact uniaxial compression to 50%, 60%, 70%, or 80% strain at a rate of 100% strain/s. Other explants were left uninjured (control specimens). All specimens underwent a culture process for 28 days and were subsequently evaluated histologically for characteristics of injury and early stages of osteoarthritis, including articular surface damage, chondrocyte cell death, focal cell loss, chondrocyte cluster formation, and loss of the extracellular matrix molecules aggrecan and types I and II collagen. Compression to all degrees of strain induced some amount of pathological change typical of clinical osteoarthritis in horses; however, only compression to 60% strain induced significant changes morphologically and biochemically in the extracellular matrix. The threshold strain necessary to model injury in full-thickness cartilage specimens from the trochlear ridges of the distal femur of adult horses was 60% strain at a rate of 100% strain/s. This in vitro model should facilitate study of pathophysiologic changes and therapeutic interventions for osteoarthritis.

Fibroblast growth factor-2 induced chondrocyte cluster formation in experimentally wounded articular cartilage is blocked by soluble Jagged-1

Basic fibroblast growth factor (FGF2) is a mitogen for articular chondrocytes. Cell death frequently occurs upon cartilage wounding and is evident during the progression of osteoarthritis. We hypothesised that incubation of wounded articular cartilage with exogenously added FGF2 would enhance cartilage repair, replacing dead cells through increased cell proliferation. Articular cartilage from the metacarapalphalangeal joint of immature bovine steers was wounded in situ, then incubated in vitro in the continual presence or absence of FGF2. Cellular proliferation was expressed as a ratio of cell density of a fixed area between wounded and adjacent cartilage. Immunolabelling revealed the incorporation of bromodeoxyuridine and localisation of collagen type VI and Notch1 epitopes. gamma-secretase inhibitor N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester and soluble Jagged1 ligand (sJ1) were used to analyse the function of Notch signalling in this wound model. FGF2 induced cellular proliferation at the margins of wounded articular cartilage, where proliferative chondrocytes adopted a cluster configuration. Collagen type VI protein was expressed by chondrocytes in clusters, as was Notch1. Cellular proliferation was not affected by inhibition of gamma-secretase dependent Notch1 signalling. Binding of sJ1 to Notch1 receptors in FGF2 treated cartilage inhibited proliferation. Addition of FGF2 induces rapid chondrocyte proliferation in wounded cartilage, chondrocytes adopt a cluster morphology and also express Notch1. Binding of sJ1 to Notch1 causes apoptosis overriding a proliferative response. This study may shed some light on the significance of increased Notch1 expression and its localisation in chondrocyte clusters in osteoarthritic cartilage.

Chondrocytes culture in three‐dimensional porous alginate scaffolds enhanced cell proliferation, matrix synthesis and gene expression

For the limited availability of autologous chondrocytes, a cultured system for expansion in vitro until sufficient cells are obtained must be developed. These cells must maintain their chondrocyte phenotype in vitro as well as in vivo, following implantation to ensure that differentiated chondrocytes synthesize a normal hyaline cartilage matrix and not a fibro-cartilage matrix. This study uses porous three-dimensional (3-D) alginate scaffolds within a perfusion system to culture low-density (5 x 10(5) cells) primary porcine chondrocytes for 1-4 weeks to study their proliferation and differentiation. The results of RT-PCR reveal that most cells could maintain their differentiation state for up to 4 weeks of culturing. Chondrocytes proliferated to 3 x 10(7) cells after 4 weeks in culture. Alginate scaffolds induced the formation of chondrocyte clusters and stimulated the synthesis of matrix, which effects were evaluated using histology and electron microscopy. These findings demonstrate that culturing chondrocytes in alginate scaffolds may effectively prevent the dedifferentiation and improve autologous chondrocyte transplantation.